PROJECT SUMMARY/ABSTRACT The overall vision of the proposed research program is to determine how evolutionary conserved mitochondrial quality control modules function to maintain cell survival, and how these functions can be manipulated to achieve clinical benefits. Research in the Khalimonchuk lab is focused on providing a better understanding of multilayered processes that mediate mitochondrial biogenesis, function and integrity. The general goals include: 1) thorough molecular characterization of factors involved; 2) dissection of their roles and concerted action in relevant biological mechanisms; and 3) investigation of associated regulatory and signaling events and their potential therapeutic relevance. To that end, this MIRA proposal focuses on a group of fundamental mechanisms that mediate mitochondrial fidelity and preserve normal mitochondrial functions throughout a lifecycle of a cell/organism. The mitochondria and its complex protein homeostasis (proteostasis) network involve multiple dynamic processes and pathways that adapt to homeostatic challenges and change over time. The multifaceted biological roles of these mechanisms are highly relevant to both normal cellular physiology and a wide range of human disorders for which no effective therapies presently exist. Progressive mitochondrial failure has emerged as a central causative factor of prevalent degenerative diseases such as glaucoma, hearing loss, Parkinsonism, amyotrophic lateral sclerosis, various neuropathies, dementia, cardiovascular disorders, and certain cancers. In spite of much research, the molecular etiology of these pathologies remains obscure. Thus, a greater understanding of the mechanisms that mediate mitochondrial fidelity, function, and integrity, and how failure of these mechanisms drives the above clinical manifestations, is of central importance. Unified by an overarching topic of mitochondrial fidelity and protein homeostasis, this research program seeks to study: 1) the mechanisms that proteolytically control/regulate protein stability, abundance, assembly, and repair within the inner mitochondrial membrane and 2) how a delicate protein homeostasis in the mitochondrial matrix is established and maintained. In each case, the proposed work will focus on a group of dedicated versatile factors that mediate mitochondrial self-preservation and are highly relevant to human health. The goal is to address unresolved questions concerning the functional roles of key molecular factors using two lauded complementary models, the yeast Saccharomyces cerevisiae and cultured human cells. The research will uncover novel mechanisms of regulation, dynamic integration, and concerted spatiotemporal action that are critical for mitochondrial sub-proteomes to adjust to homeostatic insults and physiological demands and challenges. Successful outcomes from this project will provide unprecedented insights into the coordination of mitochondrial metabolism, bioenergetics, and proteostasis and challenge current views on mechanisms of age-associated maladies. They may also aid in the development of new treatment/prophylaxis strategies against degenerative diseases and related pathologies that occur with age.